Differentially encoded biological analyzer planar array apparatus and methods

ABSTRACT

A method of probing a plurality of analyzer molecules distributed about a detection platform is disclosed. The method includes contacting a test sample to the plurality of analyzer molecules, scanning the plurality of analyzer molecules at a rate relating to a carrier frequency signal, and detecting the presence or absence of a biological molecule based at least in part upon the presence or absence of a signal substantially at a sideband of the carrier frequency signal. A molecule detection platform including a substrate and a plurality of targets positioned about the substrate is also disclosed. Specific analyzer molecules adapted to bind a specific analyte are immobilized about a first set of the targets. Nonspecific analyzer molecules are immobilized about a second set of the targets. The targets positioned about the substrate along at least a segment of a scanning pathway alternate between at least one of the first set and at least one of the second set. A method including providing a substrate for supporting biological analyzer molecules the substrate including at least one scanning pathway is also disclosed. The scanning pathway includes a plurality of scanning targets. Specific biological analyzer molecules adapted to detect a specific target analyte are distributed about a first set of the targets which alternate in groups of at least one with a second set of the targets the second set of the targets not including the specific biological analyzer molecules.

CROSS REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication Serial No. 60/648,724, entitled “METHOD FOR CONDUCTINGCARRIER-WAVE SIDE-BAND OPTICAL ASSAYS FOR MOLECULAR RECOGNITION,” filedon Feb. 1, 2005 and the same is expressly incorporated herein byreference.

This invention was made with government support under grant referencenumber NSF ECS-0200424 awarded by the National Science Foundation. TheGovernment has certain rights in the invention.

TECHNICAL FIELD

The present invention generally relates to apparatus, methods andsystems for detecting the presence of one or more target analytes orspecific biological materials in a sample, and more particularly to alaser compact disc system for detecting the presence of biologicalmaterials and/or analyte molecules bound to target receptors on a discby sensing changes in the optical characteristics of a probe beamreflected, transmitted, or diffracted by the disc caused by thematerials and/or analytes.

BACKGROUND

In many chemical, biological, medical, and diagnostic applications, itis desirable to detect the presence of specific molecular structures ina sample. Many molecular structures such as cells, viruses, bacteria,toxins, peptides, DNA fragments, pathogens, and antibodies arerecognized by particular receptors. Biochemical technologies includinggene chips, immunological chips, and DNA arrays for detecting geneexpression patterns in cancer cells, exploit the interaction betweenthese molecular structures and the receptors. [For examples see thedescriptions in the following articles: Sanders, G. H. W. and A. Manz,Chip-based microsystems for genomic and proteomic analysis. Trends inAnal. Chem., 2000, Vol. 19(6), p. 364-378. Wang, J., From DNA biosensorsto gene chips. Nucl. Acids Res., 2000, Vol. 28(16), p. 3011-3016;Hagman, M., Doing immunology on a chip. Science, 2000, Vol. 290, p.82-83; Marx, J., DNA Arrays reveal cancer in its many forms. Science,2000, Vol. 289, p. 1670-1672]. These technologies generally employ astationary chip prepared to include the desired receptors (those whichinteract with the target analyte or molecular structure under test).Since the receptor areas can be quite small, chips may be produced whichtest for a plurality of analytes. Ideally, many thousand bindingreceptors are provided to provide a complete assay. When the receptorsare exposed to a biological sample, only a few may bind a specificprotein or pathogen. Ideally, these receptor sites are identified in asshort a time as possible.

One such technology for screening for a plurality of molecularstructures is the so-called immunological compact disk, which simplyincludes an antibody microarray. [For examples see the descriptions inthe following articles: Ekins, R., F. Chu, and E. Biggart, Developmentof microspot multi-analyte ratiometric immunoassay using dualflourescent-labelled antibodies. Anal. Chim. Acta, 1989, Vol. 227, p.73-96; Ekins, R. and F. W. Chu, Multianalyte microspotimmunoassay—Microanalytical “compact Disk” of the future. Clin. Chem.,1991, Vol. 37(11), p. 1955-1967; Ekins, R., Ligand assays: fromelectrophoresis to miniaturized microarrays. Clin. Chem., 1998, Vol.44(9), p. 2015-2030]. Conventional fluorescence detection is employed tosense the presence in the microarray of the molecular structures undertest. Other approaches to immunological assays employ traditionalMach-Zender interferometers that include waveguides and gratingcouplers. [For examples see the descriptions in the following articles:Gao, H., et al., Immunosensing with photo-immobilized immunoreagents onplanar optical wave guides. Biosensors and Bioelectronics, 1995, Vol.10, p. 317-328; Maisenholder, B., et al., A GaAs/AlGaAs-basedrefractometer platform for integrated optical sensing applications.Sensors and Actuators B, 1997, Vol. 38-39, p. 324-329; Kunz, R. E.,Miniature integrated optical modules for chemical and biochemicalsensing. Sensors and Actuators B, 1997, Vol. 38-39, p. 13-28;Dübendorfer, J. and R. E. Kunz, Reference pads for miniature integratedoptical sensors. Sensors and Actuators B, 1997 Vol. 38-39, p. 116-121;Brecht, A. and G. Gauglitz, recent developments in optical transducersfor chemical or biochemical applications. Sensors and Actuators B, 1997,Vol. 38-39, p. 1-7]. Interferometric optical biosensors have theintrinsic advantage of interferometric sensitivity, but are oftencharacterized by large surface areas per element, long interactionlengths, or complicated resonance structures. They also can besusceptible to phase drift from thermal and mechanical effects. Currentpractice is to perform long time integrations (as in fluorescencedetection) to achieve a significant signal. However, the longintegration times place the measurement firmly in the range of 1/f noise(frequency=1/τ, where τ is the measurement time). Likewise, SPRmeasurement approaches (for example systems from Biacore) or resonantmirror approaches (for example systems from SRU Biosystems) are angleresolved or wavelength resolved, requiring detailed measurements thattake long integration times.

While the abovementioned techniques have proven useful for producing andreading assay information within the chemical, biological, medical anddiagnostic application industries, developing improved fabrication andreading techniques for planar arrays with significant improvement inperformance over existing planar array technology is desirable.

SUMMARY

One embodiment according to the present invention includes a method ofprobing a plurality of analyzer molecules distributed about a detectionplatform. The method includes contacting a test sample to the pluralityof analyzer molecules, scanning the plurality of analyzer molecules at arate relating to a carrier frequency signal, and detecting the presenceor absence of a biological molecule based at least in part upon thepresence or absence of a signal substantially at a sideband of thecarrier frequency signal.

Another embodiment according to the present invention includes amolecule detection platform including a substrate and a plurality oftargets positioned about the substrate. Specific analyzer moleculesadapted to bind a specific analyte are immobilized about a first set ofthe targets. Nonspecific analyzer molecules are immobilized about asecond set of the targets. The targets positioned about the substratealong at least a segment of a scanning pathway alternate between atleast one of the first set and at least one of the second set.

A further embodiment according to the present invention includes amethod including providing a substrate for supporting biologicalanalyzer molecules. The substrate includes at least one scanningpathway. The scanning pathway including a plurality of scanning targets.The method further includes distributing specific biological analyzermolecules adapted to detect a specific target analyte about a first setof the targets which alternate in groups of at least one with a secondset of the targets. The second set of the targets does not include thespecific biological analyzer molecules.

Additional embodiments, aspects, and advantages of the present inventionwill be apparent from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph of noise power density versus frequency accordingto an embodiment of the present invention;

FIG. 2 shows a graph of power spectrum versus frequency according to anembodiment of the present invention;

FIG. 3 shows a distribution of elements according to an embodiment ofthe present invention;

FIG. 4 shows a distribution of elements according to an embodiment ofthe present invention;

FIG. 5 shows scanning of an element according to an embodiment of thepresent invention;

FIG. 6 shows a distribution of elements according to an embodiment ofthe present invention;

FIG. 7 shows a distribution of elements according to an embodiment ofthe present invention;

FIG. 8 shows a bio-CD according to an embodiment of the presentinvention;

FIG. 9A shows a bio-CD according to an embodiment of the presentinvention;

FIG. 9B shows a bio-CD according to an embodiment of the presentinvention;

FIG. 10A shows a bio-CD according to an embodiment of the presentinvention;

FIG. 10B shows a bio-CD according to an embodiment of the presentinvention;

FIG. 11 shows a bio-CD according to an embodiment of the presentinvention;

FIG. 12 shows scanning of elements according to an embodiment of thepresent invention;

FIG. 13 shows a detection system according to an embodiment of thepresent invention;

FIG. 14 shows a graph of time domain results of scanning adifferentially encoded MD-class calibration disk;

FIG. 15 shows a graph of frequency domain results of scanning adifferentially encoded MD-class calibration disk;

FIG. 16 shows a graph of frequency domain results of scanning adifferentially encoded MD-class disk;

FIG. 17 shows a graph of frequency domain results of scanning adifferentially encoded MD-class disk;

FIG. 18 shows a graph of frequency domain results of scanning adifferentially encoded MD-class disk;

FIG. 19 shows a graph of frequency domain results of scanning adifferentially encoded MD-class disk;

FIG. 20 shows a portion of an MD-class disk;

FIG. 21 shows a graph of time domain results of scanning the disk ofFIG. 20;

FIG. 22 shows a graph of frequency domain results of scanning the diskof FIG. 20;

FIG. 23 shows a graph of time domain results of scanning a PC-classdisk;

FIG. 24 shows a portion of a PC-class disk;

FIG. 25 shows a magnified view of a portion of FIG. 24;

FIG. 26 shows Fourier domain results of scanning the disk of FIG. 24;

FIG. 27 shows a demodulated image of the of the Fourier domain resultsof FIG. 26;

FIG. 28 shows a graph of a comparison of prescan subtraction withoutdemodulation and prescan subtraction with demodulation.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of theinvention, reference will now be made to the embodiments illustrated inthe drawings and specific language will be used to describe the same. Itwill nevertheless be understood that no limitation of the scope of theinvention is thereby intended, such alterations and furthermodifications in the illustrated device, and such further applicationsof the principles of the invention as illustrated therein beingcontemplated as would normally occur to one skilled in the art to whichthe invention relates.

With reference to FIG. 1 there is shown graph 1000 with frequencyincreasing along its x axis as indicated by x axis arrow 1020 and noisepower density increasing along its y axis as indicated by its y axisarrow 1010. Frequency can be either temporal frequency (Hz) or spatialfrequency (1/cm). Graph 1000 illustrates noise power density versusfrequency in the absence of a carrier frequency. Curve 1030 illustratesthe noise power density of total noise as it varies with frequency.Curve 1040 illustrates the noise power density of 1/f noise as it varieswith frequency. A bandwidth between frequencies 1060 and 1070 isindicated by arrows BW. The total noise for this bandwidth is given bythe area under curve 1030 labeled 1080 which represents detected noisepower for a measurement taken at bandwidth BW. The frequency range whereonly static is detectable is illustrated by arrows ST. The frequencyvalue of the 1/f noise knee is illustrated by line 1050 and representsthe frequency above which a signal may be detected over noise.

With reference to FIG. 2 there is shown graph 2000 with frequencyincreasing along its x axis as indicated by x axis arrow 2020 and powerspectrum increasing along its y axis as indicated by y axis arrow 2010.The power level of 1/f noise is illustrated by curve 2030. A DC sidebandsignal 2040 having DC sideband center frequency 2041, a carrier signal2060 having carrier center frequency 2061, and carrier sidebands 2050and 2070 having carrier sideband center frequencies 2051 and 2071,respectively, are also shown.

Graph 2000 illustrates one example of frequency domain detection of themolecular, cellular, or particulate content of a liquid or air sample inwhich an analyte binds on or in a support material to produce aperiodic, quasi-periodic or harmonic modulation of phase or amplitude ofan electromagnetic wave that probes the support material. The periodicor quasi-periodic modulation can be in time or space, leading to atime-domain carrier frequency or a space-domain carrier frequency, byrelative motion of the probe beam and support. The presence of the boundanalyte appears as a modulation sideband of the carrier frequency. Asshown in graph 2000, carrier sideband signals 2050 and 2070 indicate thepresence of one or more target analytes bound to analyzer moleculesdistributed about a support material which is probed with anelectromagnetic wave in a detection system. The detection systempreferably includes a photodetector, or another detector responsive toelectromagnetic waves, that outputs a current as described below byEquation 1:${i(t)} = {\frac{1}{2}\left( {1 + {\cos\quad\omega_{c}t}} \right)\left( {1 + {A\quad\cos\quad\omega_{m}t}} \right)}$Equation 1 has a harmonic decomposition described by Equation 2:$\begin{matrix}{{i(t)} = {\frac{1}{2} + {\frac{1}{2}\cos\quad\omega_{c}t} + {\frac{A}{2}\cos\quad\omega_{m}t} + {\frac{A}{4}{\cos\left( {\omega_{c} + \omega_{m}} \right)}t} +}} \\{\frac{A}{4}{\cos\left( {\omega_{c} - \omega_{m}} \right)}t}\end{matrix}$Equation 2 describes a DC sideband at ω_(m), a carrier band at ω_(c),and two carrier sidebands at ω_(c) −ω_(m) and ω_(c)+ω_(m) whichcorrespond to DC sideband 2040, a carrier 2060, and sidebands 2050 and2070 as shown in graph 2000. In Equations 1 and 2, t is time, i(t) isdetector output current as a function of time, ω_(c) is carrier angularfrequency, ω_(m) the modulation angular frequency, and A is the envelopeamplitude. In further embodiments detector output could be a voltage,another electrical signal, an optical signal, or a magnetic signal, forexample, or some combination of these and/or other outputs.

With reference to FIG. 3 there is shown a distribution of elements 3000including elements 3010 and 3020. Elements 3010 and 3020 are distributedabout reading pathway 3004 which is defined on a substrate. As shown bydashed lines 3030, 3040, 3050, 3060, and 3070, elements 3010 and 3020are arranged in alternating groups of four. As shown by ellipses 3006and 3008 this pattern can continue beyond the segment illustrated inFIG. 3 with the groups of four elements alternating as described above.A unit cell includes a group of four elements 3010 and a group of fourelements 3020 as is indicated by arrow UC between dashed lines 3030 and3050. Scanning footprint SF travels along reading pathway 3004 to scanthe distribution of elements 3000. Additional embodiments includealternating groups of different numbers, for example, one, two, three,five or more, and corresponding different sizes of unit cells.

Elements 3010 include specific analyzer molecules which selectively bindwith a target analyte and elements 3020 include nonspecific analyzermolecules which do not selectively bind with a target analyte but mayexhibit similar binding properties with respect to other molecules. In apreferred embodiment according to the present invention, elements 3010include specific antibodies immobilized about their surfaces, forexample, as a monolayer, fractional monolayer, partial monolayer, ornear monolayer, and elements 3020 include similarly immobilizednonspecific antibodies. For example, if an assay is to be conducted toidentify a particular mouse protein the specific antibody could be goatanti-mouse IgG (the antibody to the mouse protein produced by a goat)and the nonspecific antibody could be goat anti-rat IgG (the antibody toan analogous rat protein produced by a goat). The goat anti-mouse IgGwill selectively bind the mouse protein while the goat anti-rat IgG willnot bind with it or will have a substantially lesser binding affinity,however, both IgGs exhibit similar nonspecific background binding withmolecules other than the target analyte. In additional embodiments thenon-specific protein could be a non-IgG, for example, casein or bovineserum albumin (BSA). These proteins could be used to test generalprotein-protein background, and could be used to test for systematicsthat are common to both groups of immobilized molecules. In furtherembodiments the specific analyzer molecules could be a cDNA that iscomplimentary to the target DNA, and the non-specific group could be astatistically similar, but not identical, cDNA. Additional embodimentscal include specific and non-specific aptamers. A variety of otherspecific and nonspecific antibody pairs may also be used, includingthose exhibiting varying degrees of similarity in nonspecific backgroundbinding and those not exhibiting similar nonspecific background binding.Furthermore, combinations of specific and nonspecific analyzer moleculesother than antibodies may also be used. Additionally, nonspecificanalyzer molecules may be omitted entirely in which case elements 3020would not include immobilized molecules. These alternative exemplaryembodiments and others can be used in connection with the presentembodiment and also in connection with the other embodiments includingthose described elsewhere herein.

Distribution of elements 3000 is one example of differential encoding orenvelope modulation of bimolecular information. According to a preferredembodiment of the present invention, distribution of elements 3000 is ona bio-CD where elements 3010 and 3020 are interferometricmicrostructures formed on a surface of the bio-CD, and reading pathway3004 is one of a number of a substantially concentric circular tracks.As described above, elements 3010 on the track are active (carrying aspecific biological analyzer molecule) and elements 3020 are inactive(carrying nonspecific molecules, no molecules, or inert molecules thatmay be comparable in size with the analyzer molecule). In this 4 on/4off format, the carrier frequency corresponds to the positioning of eachindividual one of elements 3010 and 3020, and the detection frequencycorresponds to the repeat period of the unit cell UC which is everyeight elements. Thus, the detection frequency is equal to one-eighth ofthe carrier frequency. At disk rotation speeds of 6000 rpm (100 Hz) and1024 elements per track, the carrier frequency is approximately 100 kHzand the detection frequency is approximately 12.5 kHz. A wide variety ofother bimolecular platforms, scanning rates, and element distributionsincluding, for example, those described herein, are contemplated and canresult in a variety of other carrier frequencies and detectionfrequencies.

According to a preferred embodiment of the present invention, an opticaldetection system including two phase-locked loops in series, with thefront end referenced to the carrier frequency, and the back endreferenced to the unit cell can be used to scan a bio-CD havingdistribution of elements 3000 with a laser. Differential encoding ofdistribution of elements 3000, for example as described above andelsewhere herein, can preferably reduce susceptibility to laserintensity drift or disk wobble by subtracting out these and other systemdrifts and biases, and can preferably directly subtract non-specificbackground binding, for example if the off region is printed withnonspecific antibody. One example of a detection system according to apreferred embodiment of the present invention can be found in U.S. Pat.No. 6,685,885 which is hereby incorporated by reference. This detectionsystem could also be any other detection system responsive toelectromagnetic waves including for example those described elsewhereherein.

According to a preferred embodiment of the present invention thedetection system can utilize phase quadrature interferometrictechniques. Examples of phase quadrature interferometric techniquesinclude the micro-diffraction quadrature class (“MD-class”) and adaptiveoptic quadrature class (“AO-class”) as described in U.S. applicationSer. No. 10/726,772 filed on Dec. 3, 2003 entitled “AdaptiveInterferometric Multi-Analyte High-Speed Biosensor” (published on Aug.26, 2004 as U.S. Pub. No. 2004/0166593), the contents of which areincorporated herein by reference. Other examples of phase quadratureinterferometric techniques include the phase-contrast quadrature class(“PC-class”) as described in U.S. Provisional Patent Application No.60/649,070, filed Feb. 1, 2005, entitled “Phase-Contrast Quadrature ForSpinning Disk Interferometry And Immunological Assay”, U.S. ProvisionalPatent Application No. 60/755,177, filed Dec. 30, 2005, entitled“Phase-Contrast BioCD: High-Speed Immunoassays at Sub-Picogram DetectionLevels”, and U.S. Application Serial No. ______ being filed the same dayas the present application that claims priority to these two provisionalapplications and entitled “Method And Apparatus For Phase ContrastQuadrature Interferometric Detection Of An Immunoassay.” The disclosureof the utility application being filed on the same day as the presentapplication is incorporated herein by reference. Additionally, furtherembodiments of the present invention include detection systems adaptedto utilize surface plasmon resonance or SPR, fluorescence, resonance andother techniques in which high frequency modulation in time or spaceoriginates from analyte bound to a solid support with a spatialfrequency that is scanned to produce a sideband indicating the presenceof the analyte. Still other preferred embodiments of the presentinvention include detection platforms for use in these and otherdetection systems which include distributions of targets includinganalyzer molecules which produce sideband signals that depend uponmodulation indicative of the presence of an analyte.

With reference to FIG. 4 there is shown a biosensor platform 4000including a substrate 4030 having an upper surface 4010 and lowersurface 4020. Interferometric elements 4040, 4050, 4060, and 4070 areformed on the upper surface 4010 of substrate 4030. Platform 4000 mayalso include additional interferometric elements in addition to thoseshown in the portion of platform 4000 illustrated in FIG. 4. A laserbeam 4002 having wavelength λ scans the interferometric elements 4040,4050, 4060, and 4070 in the direction indicated by arrow DM. Elements4040 and 4050 include specific analyzer molecules immobilized abouttheir scanned surfaces and elements 4060 and 4070 include nonspecificanalyzer molecules immobilized about their scanned surfaces. Thesespecific and nonspecific analyzer molecules can be, for example, thesame or similar to those described above in connection with FIG. 3 andelsewhere herein. This configuration of specific and nonspecificanalyzer molecules of biosensor platform 4000 is another example ofdifferential encoding according to a preferred embodiment of the presentinvention. In one preferred embodiment of the present invention platform4000 is a micro-diffraction bio-CD and elements 4040, 4050, 4060, and4070 are radial spokes distributed about the surface of the bio-CD.Platform 4000 can also be any of various other biosensor platformsincluding, for example, those described herein.

Biosensor platform 4000 is one example of carrier suppression accordingto a preferred embodiment of the present invention. Elements 4060 and4040 have a height illustrated by arrows HA and elements 4050 and 4070have a height illustrated by arrows HB. Height HA is about λ/8 andheight HB is about 3λ/8. Successive scanning of elements alternatingbetween height HA and HB flips the phase quadratures detected forsuccessive elements. This results in a modulation at about twice theamplitude as compared to a platform having interferometric elements withsubstantially uniform element heights. The carrier is suppressed by anapproximately π phase difference between phase quadrature signalsdetected for successive elements. Carrier suppression may be useful in avariety of circumstances. In one example, where carrier side bands areweak relative to the carrier, carrier noise can impact detection. Inanother example where carrier sidebands overlap with the carrier,carrier noise can also impact detection. Carrier wave suppression canpreferably increase the ratio of signal to noise. Complete carriersuppression or double sideband detection may be used to improve thesignal to noise ratio of detection in these and other situations.Partial carrier suppression may also improve the signal to noise ratioof detection in these and other situations. Carrier wave suppression canalso be accomplished in other manners, for example, fabrication of diskstructures and reflectivities relative to beam width, through use of aclipper circuit that clips the high signal detected from a land of adetection platform, or through use of a filter, for example a band stopfilter.

With reference to FIG. 5 there is shown an example of a scanning 5000during which footprint 5020 passes over element 5010. Areas 5021 are theareas of the scanning footprint not over element 5010 and area 5011 isthe area in which scanning footprint 5020 overlaps element 5010.According to a preferred embodiment element 5010 is a goldmicrodiffraction element placed on a partially reflecting substrate.This embodiment allows carrier suppression by the total power reflectedfrom the element being equal to the total power reflected under thecondition of quadrature which removes the large modulation caused by theapproximately 50% amplitude modulation of a micro diffraction bio-CD.This effect can be illustrated through the following equations. Thetotal electrical (far) field is given by Equation 3:$E_{T} = {\frac{E_{0}}{\sqrt{A}}\left\lbrack {{r_{L}A_{L}} + {r_{r}A_{r}{\mathbb{e}}^{i\quad\Phi}}} \right\rbrack}$The total reflected intensity is given by Equation 4:$I_{T} = {\frac{E_{0}^{2}}{A}\left\lbrack {{R_{L}A_{L}^{2}} + {R_{r}A_{r}^{2}} + {2r_{L}r_{r}A_{L}A_{r}\cos\quad\phi}} \right\rbrack}$Under the condition of Land: Φ=0, A_(L)=A and A_(r)=0. Thus, intensityreflected by land is given by Equation 5:I _(L) =I ₀ R _(L)Under the condition of Quadrature: Φ=π/2. Thus, the reflected intensityunder a condition of quadrature is given by Equation 6: $\begin{matrix}{I_{Q} = {\frac{E_{0}^{2}}{A}\left\lbrack {{R_{L}A_{L}^{2}} + {R_{r}A_{r}^{2}}} \right\rbrack}} \\{= {I_{0}\left\lbrack {{R_{L}a_{L}^{2}} + {R_{r}a_{r}^{2}}} \right\rbrack}}\end{matrix}$where a_(i) is the area fraction, and a_(L)+a_(r)=1. Conditions ofbalanced operation are given by Equations 7 and 8:I _(Q) = _(L)R _(L)α_(L) ² +R _(r)α_(r) ² =R _(L)The solution of which are given in Equations 9 and 10:$\frac{1 - a_{L}}{1 + a_{L}} = \frac{R_{L}}{R_{r}}$$a_{L} = \frac{1 - \frac{R_{L}}{R_{r}}}{1 + \frac{R_{L}}{R_{r}}}$For Equations 3-10, I_(r) is the total reflected intensity, I_(L) is theintensity reflected by land, I_(O) is the incident reflected intensity,I_(Q) is the reflected intensity under a condition of quadrature, E_(o)is the reflected field, A is the total area, A_(L) is area 5021, A_(r)is area 5011, a_(L) is A_(L) divided by the area of the beam footprint,a_(R) is A_(L) divided by the area of the element 5010 intersectingelement 5020, R_(L) is |r_(L)|², R_(r) is |r_(r)|² and Φ is the phasedifference between reflected components of the laser. Thus, if thepartially reflective substrate is silicon, for example, which hasR_(L)=32% and R_(r)=98%, then a_(L)=51% and a_(r)=49%.

With reference to FIG. 6 there is shown a biosensor platform 6000including substrate 6030 having an upper surface 6010 and a lowersurface 6020. Upper surface 6010 includes analyzer molecules 6040, 6050,6060, 6070, 6080 and 6090 immobilized about surface 6010. Analyzermolecules 6040, 6060, and 6080 are specific analyzer molecules forselectively binding a particular analyte and analyzer molecules 6050,6070 and 6090 are nonspecific analyzer molecules. The specific andnonspecific analyzer molecules can be, for example, the same or similarto those described elsewhere herein. FIG. 6 shows one example of analternating pattern of specific and nonspecific analyzer molecules.Laser beam 6002 scans the analyzer molecules in the direction indicatedby the arrow DM which is preferably accomplished by rotating theplatform 6000 but could also be accomplished by other movement ofplatform 6000 or by movement of beam 6002. According to a preferredembodiment of the present invention platform 6000 is a phase contrastbio-CD or an adaptive optical bio-CD and analyzer molecules 6040, 6050,6060, 6070, 6080 and 6090 are radial spokes or other patterns ofanalyzer molecules, however, platform 6000 could also be another kind ofbio-CD or other platform including, for example, those describedelsewhere herein.

During scanning of platform 6000 by laser beam 6002 signal phasemodulation depends only upon the binding differences between thespecific and nonspecific analyzer molecules. For example, nonspecificbinding that is common to both the types of analyzer molecules is notimparted onto the signal beam or has minimal impact on the signal beam.The detected signal is therefore independent of nonspecific binding. Inthis embodiment there is no signal detected at or about the carrierfrequency and only the modulation caused by binding of the specificanalyte and the specific analyzer molecule is detected. This is oneexample of differential encoding including carrier wave suppression anddouble sideband detection.

With reference to FIG. 7 there is shown a biological analyzer platform7000 including substrate 7030 including upper surface 7010 and lowersurface 7020. Interferometric elements 7070 are distributed about uppersurface 7010 and are spaced apart by gaps 7060. Interferometric elements7070 include specific biological analyzer molecules 7040 and nonspecificbiological analyzer molecules 7050 immobilized about their surfaceswhich can be the same or similar to those described elsewhere herein.Groups of the interferometric elements and analyzer molecules 7090 and7091 are also shown. Groups 7090 and 7091 have patterns of specific andnonspecific analyzer molecules that are at spatial frequencies with a πphase difference, that is, the positions of specific and nonspecificanalyzer molecules are flipped between groups 7090 and 7091. Platform7000 is preferably an adaptive optical bio-CD, however, platform 6000could also be any other type of biosensor platform or another type ofbio-CD including, for example, those described elsewhere herein.

During scanning of platform 7000 by a laser beam the phase of thecarrier is periodically flipped by π for successive groups 7090 and7091. The effect of the phase flipping of the carrier is that thecarrier is suppressed in the power spectrum and the modulation due tobinding of a specific analyzer molecule to the specific antibodies isdetectable at carrier sidebands. This is one example of differentialencoding including carrier wave suppression and double sidebanddetection.

According to a preferred embodiment modulated signals are detectedwithin a detection bandwidth Δf_(d). Narrow bandwidths reject morenoise, but the detection bandwidth should preferably not be smaller thanthe signal bandwidth, otherwise a part of the signal is rejected withthe noise. The signal bandwidth is determined by the relationshipdescribed by Equation 11:Δω₈Δτ=1where Δω_(s)=2πΔf_(s), Δf_(s) is the signal bandwidth, and Δτ is theduration of either a contiguous part of the signal, or the duration ofthe signal detection measurement. In preferred embodiments utilizingbio-CDs, the carrier frequency, f_(carrier), is set by the rotationfrequency of the bio-CD, f_(disk), and by the number of spokes, targets,or interferometric elements, N, around a specified circumference asdescribed by Equation 12:ƒ_(carrier) Nƒ _(disk)The signal bandwidth Δƒ_(s) is described by Equation 13:${\Delta\quad f_{s}} = \frac{f_{disk}}{2\pi}$The relative signal bandwidth Δƒ_(rel) is described by Equation 14:${\Delta\quad f_{rel}} = \frac{\Delta\quad f}{f_{carrier}}$For a single continuous track around a circumference, the relativebandwidth Δƒ_(rel) is described by Equation 14:${\Delta\quad f_{rel}} = {\frac{1}{2{\pi\Delta\tau}\quad f_{carrier}} = {\frac{f_{disk}}{2\pi\quad{Nf}_{disk}} = \frac{1}{2\pi\quad N}}}$If a circumference is divided into S equal arcs of M spokes, therelative bandwidth increases by a factor of S as described by Equation15:${\Delta\quad f_{rel}^{S}} = {{\frac{N}{M}\Delta\quad f_{rel}} = {S\quad\Delta\quad f_{rel}}}$Thus, for example, if N=1024, and S=16, the relative bandwidth is 0.25%.If ƒ_(disk)=100 Hz, then ƒ_(s)=100 kHz, Δƒ_(s)=16 Hz and Δƒ_(s rel)=256Hz. These relations suggest that S up to 128 segments or more is clearlya possible scenario for homogeneous bandwidths for which Δƒ_(s)=2 kHzand Δƒ_(s rel)=2%.

The foregoing example describes the case of homogeneous signalbandwidth. Signal bandwidths in practice are generally larger than thehomogeneous bandwidths. These arise, for example, from frequencyinstability, which in the bio-CDs is from inhomogeneities in thefabricated or printed spokes. If the placement of the spokes is onlyaccurate to 10 microns, then the bandwidth of the repetitive spokepattern is approximately 4 kHz with a relative bandwidth of 4%. Thisinhomogeneous signal bandwidth sets the correct detection bandwidth forthe bio-CDs. The number of segments can be increased to increase thehomogeneous bandwidth until it is equal to the inhomogeneous bandwidthto the relationships described by Equations 16 and 17:Δƒ^(S)=Δƒ_(in hom)BW=√{square root over (2)}ƒ _(in hom)For detection bandwidth BW, this sets the maximum segment numberaccording to Equation 18:$S = {\sqrt{2}\pi\quad{N\left( \frac{BW}{f_{carrier}} \right)}}$which for BW=3 kHz and f_(carrier)=100 kHz for N=1024, this sets themaximum S=136.

The ability to support segments suggests a disk array layout thatsegments the printed antibodies into wells. For N wells on a disk or Ssegments, the size of a well and its radial thickness are given byEquations 19 and 20:$a = {{r\quad d\quad\theta\quad{dr}} = {{r\quad\frac{2\pi}{S}{dr}} = {A/N}}}$${dr} = {\frac{AS}{2\pi\quad{rN}} = \frac{\left( {R_{2}^{2} - R_{1}^{2}} \right)S}{2\quad{rN}}}$where a is the area of a well, r is radius, dr is radial thickness of awell, θ is angular position, dθ is well arclength, A is the area of theannular region between radii R₂ and R₁, N is number of wells, S is thenumber of segments, R₁ is the inner radius, and R₂ is the outer radius.

With reference to FIG. 8 there is shown a bio-CD 8000 according to oneembodiment of the present invention. Bio-CD 8000 is a 100 mm diameterdisk or silicon wafer, however, any other dimension disk, wafer chip orother substrate or platform could also be used. Bio-CD 8000 includessectors 8001, 8002, 8003, 8004, 8005, 8006, 8007, 8008, 8009, 8010,8011, 8012, 8013, 8014, 8015, and 8016. Bio-CD 8000 further includessubstantially concentric tracks of wells 8021, 8022, 8023, 8024, 8025,8026, 8027, and 8028. Bio-CD 8000 has S=16 sectors, N=128 then T=8(tracks) and the inner track radius and radial thicknesses are given inTable I: Inner Track Radius Radial Thickness dr Track Number(millimeters) (millimeters) 8028 20 6.56 8027 26.56 4.94 8026 31.50 3.608025 35.10 3.39 8024 38.50 3.13 8023 41.63 2.93 8022 44.56 2.76 802147.33 2.62Bio-CD 8000 is one example of an equal area well layout according to thepresent invention. Other layouts are also contemplated, for example, a512 well layout with S=16, T=32, and any other combination of sectorsand tracks. According to a preferred embodiment layouts are used whichbring the aspect ratio of arclength and radial thickness closer to unitywhich simplifies fabrication. Fabrication of this and other embodimentsof the present invention can include particular features for variousclasses of bio-CDs. For example, a micro-diffraction bio-CD can haveradial spokes fabricated from gold across the entire disk, and wellsdefined by hydrophobic dams. A pin plotter or ink-jet printer modifiedfrom biochip array printers can be used to deposit an equal amount ofanalyzer molecules into each well. Different antibodies can be depositedwhich then self-immobilize on thiolated gold. In another example gelprinting can be used. In another example, for adaptive optical bio-CDsand phase constant bio-CDs, spokes can be printed as inert protein, damscan be put into place and antibody deposited into the wells by pin arrayplotters or protein spotters.

With reference to FIGS. 9A, 9B, 10A, 10B and 11 there are shown bio-CDs9000A, 9000B, 10000A, 10000B, and 11000 according to embodiments of thepresent invention where the wells are of equal area. In theseembodiments, dr is held constant among the tracks, and ds=rdθ is alsoheld constant. This leads to a varying dθ across the disk. In thepreferred embodiment where well areas remain are equal, the radial widthof each well is constant which simplifies design of the protein plotter,and optimal use of real-estate is made. This embodiment requires acarrier spoke number C to vary with radius, also causing the carrierfrequency to vary with radius (for constant angular velocity). Therelation of the spoke number is given by Equation 21:$C = \frac{2\pi\quad r}{\Lambda}$where Λ is the spatial period, usually Λ=2w, where w is the beam waist.For a beam waist of 20 microns and Λ=40 microns, this gives the numberof spokes as a function of radius C=3000 at r=20 mm and C=8000 at r=50mm. The carrier frequencies are 300 kHz and 800 kHz, respectively.

For N wells, the area of each well is given by Equation 22:α=rdθdr=A/NThe aspect ratio a_(r) is set by the Equation 23:rdθ=α _(r) drThe radial widths and angular widths are given by Equation 24:${dr} = {\sqrt{\frac{A}{a_{r}N}}\quad{and}}$${d\quad\theta} = {\frac{1}{r}\sqrt{\frac{a_{r}A}{N}}}$FIG. 9A shows a 96 well disk with an aspect ratio of 1 and dr=7.5 mm,a=61 mm², T=4, S_(i)=15, and S_(o)=33. FIG. 9B shown a 96 well disk withan aspect ratio of 4 and dr=4.3 mm, a=64 mm², T=7, S_(i)=8, andS_(o)=19. The well in FIGS. 9A and 9B areas are approximately 0.6 cm².FIG. 10A shows a 512 well disk with an aspect ratio of 4, dr=1.76 mm,a=12.7 mm², T=17, S_(i)=17, and S_(o)=42, FIG. 10B shows a 1000 welldisk with an aspect ratio of 4, dr=1.25 mm, a=6.4 mm², T=24, S_(i)=24,and S_(o)=59. FIG. 11 shows an 8000 well disk with an aspect ratio of 4,dr=0.45 mm, a=0.82 mm², T=66, S_(i)=69, and S_(o)=172. A variety ofother disks with equal area wells and unequal well areas are alsocontemplated. In general, larger aspect ratios have narrower detectionbandwidth, but more tracks with smaller track pitches.

With reference to FIG. 12 there are shown examples of scanning targets12000. Targets 12000 are a periodically alternating pattern of targetsincluding specific antibodies 12010 and targets including nonspecificantibodies or not including antibodies 12020. Specific and nonspecificantibodies are being immobilized about a substrate, for example, asdescribed herein. After exposure to a sample including a specific targetanalyte, targets 12010 have the analyte bound to their analyzermolecules while targets 12020 exhibit little or no binding of thespecific analyte. The period of the alternating pattern is shown byarrows LL, and the spatial frequency of the pattern is inverselyproportional to its period as shown by Equation 25:$v_{spatial} = \frac{1}{\Lambda}$where Λ is the spatial periodicity and ν_(spatial) is the spatialfrequency.

During scanning targets 12000 are illuminated by a scanning footprintsuch as a laser spot. The scanning footprint could be, for example,focused laser spot vv which has a width w_(o) less than spatialperiodicity Λ (preferably w_(o)<<Λ) and moves relative to the targets12000 with a velocity in the direction indicated by arrow v. Under thesescanning conditions the spatial frequency ν_(spatial) is converted intotemporal frequency on the transmitted or reflected beam as described byEquation 26:ƒ=V·vwhere ƒ is the carrier frequency of phase or amplitude modulation.

The scanning footprint could also be, for example, broad area laser spotz which has a width w_(o) greater than spatial periodicity Λ (preferablyw_(o)>>Λ) and can be stationary or can move relative to the targets12000 with a velocity V in the direction indicated by arrow v. Whenlaser spot z is stationary and broadly illuminates the spatialfrequency, then the spatial frequency leads to diffraction at specificangles as described by Equation 27:$\theta = {\sin^{- 1}\left( \frac{\lambda}{\Lambda} \right)}$where λ is the illumination wavelength, and Λ is the spatial period.When laser spot z moves over to targets 12000, or targets 12000 movewith velocity V, then the diffracted orders acquire a phase modulationthat is time-periodic.

The foregoing examples illustrate how spatial frequencies on a scanningplatform, for example a chip or disk, can be converted into temporalfrequencies in a laser scanning system, and how the two types offrequencies can be combined when a laser probes more than one target onthe platform.

With reference to FIG. 13 there is shown detection system 13000 whichincludes detector 13010 and detector 13020. Detectors 13010 and 13020could be any detectors for detecting electromagnetic waves, for exampleoptical detectors. System 13000 further includes probe beam 13030 whichcan be a focused probe beam or a broad area probe beam. Probe beam 13030scans targets 13040 which move relative to beam 13030 with a relativevelocity in the direction indicated by arrow RV. The scanning targets13040 by beam 13030 results in a transmitted or reflected mode 13012 anda diffracted mode 13022. Mode 13012 is directed to detector 13010 andmode 13022 is directed to detector 13020. Reference beam 13023 isdirected to detector 13010 and reference beam 13023 is directed todetector 13020. Reference beam 13023 is preferably maintained in acondition of phase quadrature relative to the transmitted mode 13012.Reference beam 13033 is preferably maintained in a condition of phasequadrature relative to diffracted mode 13022. System 13000 also includesbeam splitters 13011 and 13021 which could also be adaptive optical beamcombiners. Having a reference wave that is in phase quadrature withdetected signal allows a small shift in the phase modulation of thesignal to linearly proportional change in detected intensity allowingsignal modulation per bound analyte molecule to be maximized. Referencebeams 13033 and 13023 can be added before photodetectors or can becombined adaptively with signals. Reference beams 13033 and 13023 canarise from a diffracted spatial mode, for example, in the case ofwavefront splitting, from free space, or from partial reflections, forexample, in the case of amplitude splitting. It is also contemplatedthat detection system 13000 could include only one or the other ofdetectors 13010 and 13020 and their related beams and modes.

Experimental demonstrations of several exemplary embodiments includingcarrier side band detection according to the present invention will nowbe described in connection with FIGS. 14-28. With reference to FIG. 14,there is shown graph 14000 with time increasing along its x axis asindicated by x axis arrow 1420 and signal intensity (voltage) increasingalong its y axis as indicated by y axis arrow 14010. Graph 14000 furthershows signal 14030 which is a voltage signal that varies with time.Signal 14030 results from the scanning of an MD-class calibration diskwhich was fabricated with 1024 gold spokes deposited radially on adielectric substrate. The average (mean) spoke height was 80 nm. Of the1024 spokes, 512 spokes were below the average height, 512 spokes wereabove the average height, and the spokes alternated between those abovethe average height and those below the average height.

Scanning the MD-class calibration disk produced signal 14030 whichincludes a series of alternating local minima 14031 and 14032corresponding to and indicating the two spoke heights. The signalintensity difference between the alternating local minima 14031 and14032 is illustrated by arrow 14040 and corresponds to a heightdifference of about 30 nm between alternating spokes. This heightdifference is representative of the height difference cause by certaintarget analytes to analyzer molecules. The signal level corresponding tothe average spoke height of about 80 nm is indicated by dashed line14050. The MD-class calibration disk thus provides a simulation of adifferential encoding scheme whereby every other alternating spokeincludes analyzer molecules that bind a target analyte and can becompared to a reference spoke. The fast relative comparison between thetwo types of spokes allows for significant noise reduction.

With reference to FIG. 15 there is shown graph 15000 with frequencyincreasing along its x axis as shown by x axis arrow 15020 and powerincreasing logarithmically along its y axis as shown by y axis arrow15010. Graph 15000 shows the frequency domain results of the scanning ofthe MD-class calibration disk described above in connection with FIG.14. Graph 15000 shows carrier signal 15030 at 200 kHz, sideband signal15031 at 100 kHz, and sideband signal 15032 at 300 kHz. Thus thesideband signals are present at half carrier frequency increments. Astrong 1/f noise peak 15040 is present at zero frequency, and asignificantly suppressed noise floor is present at the frequencies ofcarrier and sideband signals 15030, 15031 and 15032. The noisesuppression by operating at this scanning rate is over 60 dB or 3 ordersof magnitude better signal to noise ratio when compared to a staticmeasurement at DC (zero frequency). This is a fundamental advantage tohigh speed repetitive sampling according to certain embodiments of thepresent invention.

With reference to FIG. 16 there is shown graph 16000 with frequencyincreasing along its x axis as shown by x axis arrow 16020 and powerincreasing along its y axis as shown by y axis arrow 16010. Graph 16000shows an example of protein side-band detection for an MD-class diskhaving proteins (in this case antibody IgG molecules) immobilized on a1024-spoke disk with 64 segments composed of 8 elements with protein and8 elements without. This created a disk with an alternating pattern of 8gold spokes carrying protein followed by 8 bare gold spokes. Thispattern repeated for a total of 64 segments each with a total of 16elements divided into 8 with protein and 8 without. The proteins werepatterned using a polydimethylsiloxane (PDMS) stencil on the disk. Acontrol track which did not include printed protein was also included onthe disk. The results of scanning the control track are indicated bydotted line 16060 and the results of scanning a track including thepatterned protein are indicated by line 16050.

Graph 16000 shows 16030 the 1/f noise at DC and two DC sideband signals16031 and 16032. A carrier frequency signal (not shown) is present atabout 100 kHz. The presence of protein is detected as a 1/64 harmonic ofthe carrier frequency at about 1.6 kHz as shown by signal 16032 and alsoby signal 16031 at about −1.6 kHz. A second harmonic signal 16034 and16033 is also present at 1/32 the carrier frequency and is caused byslight asymmetry in the deposition of the proteins. A comparison ofprotein track signal 16050 and signal 16060 of a control trackcontaining no protein illustrates the strong effect of the protein inproducing sideband signals with a 20:1 signal to noise ratio asindicated by arrow 16040.

With reference to FIG. 17 there is shown graph 17000 with frequencyincreasing along its x axis as shown by x axis arrow 17020 and powerspectrum increasing logarithmically along its y axis as shown by y axisarrow 17010. Graph 17000 presents average values for scanning of sixtracks of the MD-class disk which is described above in connection withFIG. 16. Graph 17000 shows a comparison of 1/64 harmonic signal 17040 atabout 1.6 kHz, which is generated by and indicates the presence ofprotein, and carrier signal 17030. As illustrated by arrow 17050, theprotein modulation is about 4.6% of the carrier wave, which isconsistent with a monolayer of immobilized protein.

With reference to FIG. 18 there is shown graph 18000 with frequencyincreasing along its x axis as shown by x axis arrow 18020 and powerspectrum increasing logarithmically along its y axis as shown by y axisarrow 18010. While the side bands off of DC yielded the bestsignal-to-noise ratio for scanning the MD-class disk described above inconnection with FIG. 16, every carrier harmonic includes two side-bands.Thus, as shown in graph 18000 fundamental carrier harmonic 18030 whichis at about 80 kHz includes sidebands 18031 and 18032. Sidebands 18031and 18032 are small peaks above and below the harmonic carrier frequency18030 which indicate the presence of the protein. Every other carrierharmonic also has two associated sidebands.

With reference to FIG. 19 there is shown graph 19000 with frequencyincreasing along its x axis as shown by x axis arrow 19020 and powerspectrum increasing logarithmically along its y axis as shown by y axisarrow 19010. Graph 19000 shows carrier frequency harmonics 19030A (whichis the first carrier harmonic 18030 at about 80 kHz described above inconnection with FIG. 18), 19030B, 19030C, 19030D, 19030E, 19030F, and19030G. Each carrier harmonic includes protein sidebands, though thewide frequency range of the graph 1900 makes it difficult to see theprotein sidebands for all the harmonics. Graph 19000 also demonstratesthe noise-floor roll-off for high frequencies associated to the transittime t=w₀/v of a point on the disk across the width of the focused laserspot w_(o). Line 19050 shows the approximate midpoint of the noise floorroll off.

With reference to FIG. 20 there is shown a fluorescence microscope imageof portion of an MD-class disk 20000 according to one embodiment of thepresent invention. Disk 20000 is a half-harmonic differentially encodedMD-class disk which was created using photolithography to immobilizeprotein on every alternating spoke. During this process half the spokeswere covered by photo-patterned photoresist while the other half wereexposed to protein. The photoresist was then removed to uncover baregold spokes. This results in a disk where protein is immobilized onevery alternating spoke as shown by lines 20010 (indicating depositionof specific antibody) and 20020 (indicating no deposition of antibody,or deposition of a non-specific antibody). The width of each proteindeposit is about 20 microns as indicated by arrows SW. Thishalf-harmonic differential encoding in which every alternating spokecarries protein results in the highest signal-to-noise ratio beingattained. This provides for the highest-frequency differencingmeasurements, and also boosts the total protein signal when thezero-frequency upper sideband and the carrier frequency lower side-bandmerge into a single sideband half way between DC and the fundamentalcarrier frequency.

With reference to FIG. 21 there is shown graph 21000 with timeincreasing along its x axis as shown by x axis arrow 21020 and voltageincreasing along its y axis as shown by y axis arrow 21010. When a 512differential encoded disk is rotated and scanned, the protein modulatesthe gold spokes with a frequency at half the fundamental carrierfrequency. Graph 21000 shows the detected time trace 21030 from a 512differential encoded disk. Trace 21030 shows an alternating patternbetween the bare and protein-carrying spokes as indicated by the minimumpoints trace 21030 which alternate in amplitude at the rate of a halfharmonic signal 21040.

With reference to FIG. 22 there is shown graph 22000 with frequencyincreasing along its x axis as shown by x axis arrow 22020 and powerspectrum increasing logarithmically along its y axis as shown by y axisarrow 22010. Graph 22000 shows the frequency domain side band effect ofthe disk described above in connection with FIG. 21. The half-frequencyharmonic protein signal 22040 is strong and occurs near the frequency oflowest noise between DC signal 22050 and the first carrier signal 22030.As shown in graph 22000 the DC sideband and first carrier sidebands havemerged at the half-frequency harmonic protein signal 22040. Furthermore,the protein signal 22050 itself has sidebands 22041 and 22042 caused byslight asymmetries in the protein printing. The signal-to-noise ratio isgreatest in this situation where the noise floor is lowest. Thus,detection of protein at signal 22040 represents the optimal performancecondition for carrier sideband detection on the MD-class disk describedabove.

With reference to FIG. 23 there is shown graph 23000 with frequencyincreasing along its x axis as shown by x axis arrow 23020 and powerspectrum increasing logarithmically along its y axis as shown by y axisarrow 23010. Graph 23000 shows the power spectrum for an embodiment of aPC-class disk with a periodic pattern of protein on a dielectric diskwith no other disk structure. Graph 23000 shows DC signal 23040 andprotein signal 23030 which is caused by and indicates the presence ofprotein. For this PC-class embodiment, the carrier frequency isattributable entirely to the protein, without any contribution frommicrostructures or other physical structures on the disk. The detectionof periodic patterns of immobilized protein on a flat surface is oneexample of carrier-wave suppression that was discussed above. Additionalembodiments including, for example, suppressing the carrier of the goldspokes on MD-class disks are also discussed above. Analyzer moleculepatterns on PC-class disks offer a embodiment of side-band detection andmanipulation that significantly improves the sensitivity of the bio-CDbecause the periodic protein patterns can themselves be modulated toform larger spatial patterns.

With reference to FIG. 24 there is shown a portion of a patternedprotein PC-class disk 24000 according to one embodiment of the presentinvention. The radial direction is in the vertical direction the angulardirection around the disk is in the horizontal direction. As shown inFIG. 11, the portion of disk 24000 is in a checkerboard pattern.Substantially rectangular areas of periodic stripes of protein 24010 arealternated with substantially rectangular areas of bare disk 24020. Eachsubstantially rectangular area has a radial distance of approximately0.5 mm indicated by arrow RD and an angular distance of approximately 45degrees indicated by arrow AD. The height of the printed protein stripesis approximately 5 nm. The signal resulting from scanning the PC-classdisk is differential, showing only the steps up and down from theprotein stripes.

With reference to FIG. 25 there is shown a magnified portion 25000 ofthe PC-class disk 24000 shown in FIG. 24 individual protein bands 24011of protein regions 24010 are visible in magnified portion 25000. Therectangular spatial patterns of areas 24010 and 4020 of disk 24000create sidebands on the protein peak in the power spectrum. Thelong-range spatial patterns can be detected using a sidebanddemodulation process conceptually similar to the demodulation of FMradio. The long-range protein patterns constitute an envelope thatmodulates the carrier wave. By demodulation, the envelope is extracted.Because it is more slowly varying, envelope demodulation makes itpossible to perform more accurate prescan subtraction.

An exemplary procedure for sideband detection will now be described withreference to FIGS. 26, 27 and 28. FIG. 26 shows an isolated protein peak26030 in the power spectrum. The horizontal axis 26010 is temporalfrequency and the vertical axis 26020 is spatial frequency along theradius of the disk. The sub-peaks 26031 and 26032 represent thelong-range envelope pattern. To demodulate the signal and extract theprotein envelope, this protein peak is shifted back to DC and thenFourier-transformed back into the space domain. The resultingdemodulated image is shown in FIG. 27. Only the long-range checkerboardpattern 27000 corresponding to areas 24010 and 24020 is visible, withthe periodicity of the individual protein bands 24011 removed. Afterdemodulation, subtracting a prescan becomes much more accurate.

FIG. 28 shows graph 28000 with distribution probability on the verticalaxis and height in nm on the horizontal axis. Graph 28000 shows theresults of subtracting a prescan from a postscan before demodulation asdistribution 28010 and performing the same subtraction afterdemodulation as distribution 28020. The error in height reduces from 75pm for distribution 28010 to 20 pm for distribution 28020 withdemodulation. This increased accuracy improves surface mass sensitivityby over a factor of 3 in this example.

While the examples illustrated and described above in connection withFIGS. 14-28 have made reference to particular embodiments, for example,MD-class disks with protein attached using photolithographic techniquesand PC-class disks with printed protein, these specific embodiments aremerely exemplary and it is contemplated that differential encoding andsideband detection described above could be employed with a variety ofother embodiments according to the present invention including thosedescribed elsewhere herein.

Various embodiments according to the present invention can include avariety of biosensor platforms including those described above. Forexample, these platforms include bio-CDs such as micro-diffractionbio-CDs, adaptive-optical bio-CDs, phase-contrast bio-CDs, and others.Details relating to these various classes of bio-CDs can be found, forexample, in the aforementioned patents and patent applications. Theseplatforms further include bio-chips, immunological chips, gene chips,DNA arrays, platforms used in connection with fluorescence assays andother platforms and substrates supporting planar arrays includinganalyzer molecules including, for example, those described herein.

Various embodiments according to the present invention can include avariety of analyzer molecules useful in detecting the presence orabsence of a variety of target analytes in a solution to be tested. Forexample, these analyzer molecules can include antibodies orimmunoglobulins, antigens, DNA fragments, cDNA fragments, aptameres,peptides, proteins, and other molecules. Various embodiments accordingto the present invention can include combinations of one or more of theforegoing and other types of analyzer molecules known to a person ofordinary skill in the art arranged, for example, in a planar array.

Various embodiments according to the present invention can be used inconnection with a variety of scanning and detection techniques. Forexample, such techniques include interferometry, including surfacenormal interferometry techniques, and preferably phase quadratureinterferometry techniques where one detected optical mode differs inphase from another by about π/2 plus or minus about twenty percent or anodd integer multiple thereof, and/or self referencing interferometrytechniques where a reference wave is generated locally with respect to asignal wave so that the reference and signal waves experience commonaberrations and path length changes and thus maintain a constantrelative phase without the need for active stabilization of differentlight paths, florescence techniques and platforms, resonance techniquesand platforms, and other techniques and platforms.

As used herein terms relating to properties such as geometries, shapes,sizes, physical configurations, speeds, rates, frequencies, periods,amplitudes, include properties that are substantially or about the sameor equal to the properties described unless explicitly indicated to thecontrary.

While the invention has been illustrated and described in detail in thedrawings and foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly preferred embodiments have been shown and described and that allchanges and modifications that come within the spirit of the inventionare desired to be protected.

1. A method of probing a plurality of analyzer molecules distributedabout a detection platform comprising: contacting a test sample to theplurality of analyzer molecules; scanning the plurality of analyzermolecules at a rate relating to a carrier frequency signal; anddetecting the presence or absence of a biological molecule based atleast in part upon the presence or absence of a signal substantially ata sideband of the carrier frequency signal.
 2. The method of claim 1further comprising prescanning the plurality of analyzer moleculesbefore the contacting and improving the detecting based upon adifference between the scanning and the prescanning.
 3. The method ofclaim 1 wherein the sideband is substantially free from overlap with thecarrier frequency signal.
 4. The method of claim 1 wherein the detectingutilizes self referencing phase quadrature interferometric detection. 5.The method of claim 1 wherein the detection platform is a bio-CD.
 6. Themethod of claim 1 further comprising suppressing the carrier frequencysignal.
 7. The method of claim 1 wherein the detecting utilizesinterferometry and the scanning utilizes a laser beam.
 8. The method ofclaim 1 further comprising detecting the presence or absence of a secondbiological molecule based at least in part upon the presence or absenceof a second signal substantially at a second sideband of the carrierfrequency signal.
 9. The method of claim 1 wherein the detectingincludes detecting a harmonic signal closest to zero frequency.
 10. Themethod of claim 1 wherein the detecting includes detecting a harmonicsignal at a frequency greater than that of a harmonic signal closest tozero frequency.
 11. The method of claim 1 wherein the detecting includesdetecting a signal at or about a fundamental carrier frequency.
 12. Themethod of claim 1 wherein the detecting utilizes fluorescence detection.13. A molecule detection platform comprising a substrate and a pluralityof targets positioned about the substrate wherein specific analyzermolecules adapted to bind a specific analyte are immobilized about afirst set of the targets, and nonspecific analyzer molecules areimmobilized about a second set of the targets, and the targetspositioned about the substrate along at least a first segment of ascanning pathway alternate between at least one of the first set and atleast one of the second set.
 14. The platform of claim 13 wherein thetargets positioned about the substrate alternate along the first segmentof the scanning pathway between at least four of the first set and atleast four of the second set.
 15. The platform of claim 13 wherein theplatform is a micro diffraction bio-CD, a phase contrast bio-CD, or anadaptive optics bio-CD.
 16. The platform of claim 13 wherein thenonspecific analyzer molecules exhibit nonspecific background binding atleast substantially similar to the specific analyzer molecules.
 17. Theplatform of claim 13 wherein the targets are interferometricmicrostructures.
 18. The platform of claim 13 wherein the targetspositioned about the substrate along at least a second segment of thescanning pathway adjacent the first segment alternate between at leastone of the second set and at least one of the first set in the oppositeorder as the alternation of the first segment.
 19. The platform of claim13 wherein the targets are substantially contiguous along the segment ofa scanning pathway.
 20. A method comprising: providing a substrate forsupporting biological analyzer molecules the substrate including atleast one scanning pathway, the scanning pathway including a pluralityof scanning targets; and distributing specific biological analyzermolecules adapted to detect a specific target analyte about a first setof the targets which alternate in groups of at least one with a secondset of the targets, the second set of the targets not including thespecific biological analyzer molecules.
 21. The method of claim 20further comprising distributing nonspecific analyzer molecules about thesecond set of the targets.
 22. The method of claim 20 wherein the firstset of the targets alternate in groups of at least four with the secondset of the targets.
 23. The method of claim 20 further comprising:contacting a test sample to the molecules; scanning the plurality oftargets at a rate; and detecting the presence or absence of a biologicalmolecule based at least in part upon the presence or absence of a signalsubstantially about a frequency offset from a frequency defined by thedistribution of the targets and the scanning rate.
 24. The method ofclaim 23 wherein the detecting utilizes fluorescence.
 25. The method ofclaim 23 wherein the substrate is a surface of a bio-CD, and thedetecting utilizes phase quadrature interferometric detection